1.5.6.1 Intrinsic deregulation of early follicular development
The characteristic histological feature of PCOS ovaries is an increased number of growing follicles, with the presence of atretic cystic follicles, in an enlarged collagenous stroma (Hughesdon, 1982). A similar ovarian phenotype is found in patients with adrenal androgen excess, such as congenital adrenal hyperplasia (White and Bachega, 2012, London, 1987), indicating that androgens are essential contributors to the exaggerated follicle development. Morphological studies indicate that the dysfunctional follicular development in PCOS starts at the early gonadotrophin independent stages. Ovarian cortical biopsies from anovulatory PCOS women showed a 6 times increase in density of pre-antral follicle numbers compared to normal
ovaries, including primordial and primary follicles (Webber et al., 2003). The balance between resting and growing follicles in PCOS is clearly disturbed, but the underlying mechanisms remain poorly understood (Franks and Hardy, 2010). AMH, known to play a role in suppressing primordial follicle recruitment, has been implicated in this process. Immunostaining for AMH in cross-sections of normal and PCOS ovaries have shown that primary and transitional follicles from ovaries of anovulatory PCOS women express less AMH protein (Stubbs et al., 2005). The relative AMH-deficiency in the early stage PCOS follicles could possibly contribute to the increased follicle activation. On the other hand, studies have shown, that, when sustained in in vitro culture, PCOS follicles exhibit decreased atresia rates, and survived longer than normal follicles (Webber et al., 2007). Decreased atresia and prolonged longevity would be another explanation for the increased pool of growing follicles observed in PCOS. However, in vivo, atresia rates in PCOS ovaries were comparable with normal follicles (Maciel et al., 2004).
The endocrine cells of PCOS follicles show signs of intrinsic deregulations. Human granulosa and theca cells from normal cycling women and PCOS patients exhibit different behavior in culture, especially with regard to their gonadotrophin sensitivity and the response to insulin. Granulosa cells from anovatory PCOS women were steroidogenically more active and produced 6-10 times more estradiol in response to FSH than normal granulosa cells (Mason et al., 1994). This is probably explained by the increased expression of FSHR on PCOS granulosa cells, induced by the local androgen excess (Mason et al., 1994). PCOS theca cells showed a more than 10-fold increase in basal and LH-induced androstenedione production (the main ovarian androgen secreted in vivo) during primary culture, compared with normal theca cells (Gilling-Smith et al., 1994). Production of progesterone, 17-OH progesterone, DHEA and testosterone were also increased, in basal condition, and this was more pronounced following forskolin-stimulation, mimicking LH-induction (Nelson et al., 1999). Moreover, mRNA
expression studies in PCOS-derived theca cells demonstrated increased expression of steroidogenic enzymes CYP11A1, CYP17A1 and 17b-HSD in human theca cells, and the activity of CYP17A1, 3b-HSD and 17b-HSD was increased compared with theca cells obtained from normally cycling women (Nelson et al., 1999). Normal small pre-antral follicles were found to become responsive to LH-stimulation when they reached 9.5-10 mm diameter, while the PCOS follicles showed premature LH-responsiveness, that became obvious at diameters of only 4 mm (Willis et al., 1998). The androgen excess and the inadequate response to LH override the FSH-induced aromatase expression and prematurely switched the granulosa cell steroid outcome from oestrogen to progesterone (Willis et al., 1998).
Furthermore, 5a-reductase activity significantly increased in PCOS follicles compared with control follicles (Jakimiuk et al., 1999), and the accumulation of 5a-reduced androgen further suppressed aromatase activity (Agarwal et al., 1996).
Insulin, at physiological doses, interacts with insulin-like growth factor to increase CYP17A1 activity and androgen production in ovarian theca cells (Bergh et al., 1993). Insulin resistance, frequently associated with PCOS, results in compensatory hyperinsulinaemia, which in turn fuels the androgen hypersecretion (Diamanti-Kandarakis and Dunaif, 2012). As mentioned earlier, insulin resistance in PCOS appears to be selective. Studies in human granulosa-lutein cells, obtained at egg retrieval following gonadotrophin-stimulation in the setting of IVF, have shown that ovarian steroidogenesis in PCOS remained responsive to the stimulatory effects of insulin (Rice et al., 2005). However, the metabolic effects of insulin, i.e. glucose uptake and utilization, were decreased in granulosa-lutein cells obtained from anovulatory PCOS women (Rice et al., 2005) or even absent in PCOS women with diagnosed insulin resistance (Fedorcsak et al., 2000).
1.5.6.2 Stromal hyperplasia, rigidity, hypervascularity, and inflammation
Histological examination of polycystic ovaries revealed a highly increased thickness of the cortical, but mostly the subcortical medullar stroma, associated with hypervascularity (Hughesdon, 1982). The enlarged ovarian stromal volume can be measured by ultrasound and correlates with the degree of hyperandrogenism (Fulghesu et al., 2007, Kyei-Mensah et al., 1998). Microarray data from PCOS ovarian tissue showed differential expression of genes known to be involved in extracellular matrix organization (Jansen et al., 2004). On the protein level, several molecules involved in fibrogenesis were upregulated in PCOS stroma (Ma et al., 2007). Increased levels of basic fibroblast growth factor (bFGF) were measured in serum and follicular fluid of PCOS women (Artini et al., 2006). The enhanced fibroblast proliferation and extracellular matrix deposition results in increased rigidity of the ovarian cortex (Lebbe and Woodruff, 2013). An important research question is whether the stromal hyperrigidity contributes to the intrinsically altered steroidogenic behavior of the antral follicles in PCOS (Woodruff and Shea, 2011). Although ‘rigid’ or non-permissive culture conditions in murine in vitro follicular culture were associated with decreased steroid production in multi-layered follicle (Xu et al., 2006b), the effect of rigidity on smaller follicles has not been studied yet.
The blood flow velocity, examined by color Doppler ultrasound, was greatly increased in polycystic ovaries (Zaidi et al., 1995), mainly in the cortex (Delgado-Rosas et al., 2009). Circulating vascular endothelial growth factor (VEGF) levels were typically increased in PCOS women, and were correlated with the Doppler measurements of ovarian stromal blood flow (Agrawal et al., 1998). Laparoscopic ovarian drilling, a surgical technique aiming to reduce the ovarian volume, reduced Doppler measurements of ovarian stromal blood flow (Parsanezhad et al., 2003), with concomitant decrease of plasma levels of VEGF (El Behery et al., 2011) and androgens (Kaaijk et al., 2000). The increased cortical blood flow in PCOS maintains the
exaggerated connective tissue proliferation and, importantly, supplies the enclosed early-staged follicles with inadequate amounts of oxygen, metabolic and endocrine factors (Lebbe and Woodruff, 2013). The profoundly altered follicular microenvironment in PCOS is an important disruptor of the normal follicle dynamics (Figure 1-20).
PCOS is also characterized by a chronic low-grade inflammatory process, which is more prominent in the presence of obesity, and manifests with elevated plasma levels of C-reactive protein (CRP), inflammatory cytokines (IL-6 and others), and leucocytes (Diamanti- Kandarakis et al., 2006b). The contribution of these inflammatory parameters in the pathogenesis of PCOS remains unclear. It has been postulated that, as follicle become activated to grow, it chemically attracts stromal macrophages, which stay associated with this follicle throughout its maturation (Tingen et al., 2011). In benign prostate hypertrophy, stromal hyperplasia is partly mediated through the infiltration of macrophages, and their recruitment is described as an androgen-dependent mechanism (Izumi et al., 2013). Similar epithelial-stromal interactions might possibly play a role in the pronounced stromal hyperplasia occurring in PCOS (Lebbe and Woodruff, 2013).
The communication between the ovarian follicular and stromal compartment is a crucial notion that contributes to the complex pathogenesis of PCOS (Lebbe and Woodruff, 2013), (Figure 1-20).
Figure 1-20 Working model for follicular-stromal interactions in the pathogenesis of PCOS. Follicular arrest results from the imbalance between androgens, FSH and AMH. Circulating FSH-levels are inappropriately low and dominant follicle selection fails. The PCOS cortex shows signs of remodeling, partly mediated by increased local androgen concentrations, and maintained by increased cortical blood flow. Reprinted and adapted from Lebbe et al, with kind permission of Oxford University Press. Copyright 2013.
1.5.6.3 Oocyte quality in PCOS
The oocyte quality in PCOS has mainly been studied in the context of assisted reproduction, in infertile PCOS women not responding to ovulation-induction by clomiphene. As mentioned earlier, PCOS is characterized by an increased FSH sensitivity. During controlled ovarian stimulation, the requirement for exogenous gonadotrophins is reduced (Sahu et al., 2008, Mulders et al., 2003). To diminish the risk of ovarian hyperstimulation syndrome (OHSS) low-dose gonadotropin protocols have been developed for PCOS, facilitating the development of an appropriate amount of follicles (Kumar et al., 2011). Nevertheless, at the time of egg
retrieval, the yield is typically increased in PCOS, compared with non-PCOS controls (Heijnen et al., 2006, Weghofer et al., 2007). The fertilization potential of PCOS oocytes is decreased and indicative of compromised oocyte quality (Urman et al., 2004). The chromosomal organization and euploidy rates appear to be normal in PCOS (Sengoku et al., 1997, Weghofer et al., 2007). But the microenvironment of the oocyte, the follicular fluid, contains hallmarks of metabolic dysregulation contributing to the impaired oocyte quality, and more pronounced in obese PCOS patients, with elevated free androgen index and free fatty acids (Niu et al., 2014). The cytoplasmic oocyte development is clearly altered in PCOS, and reflected by a distinctly abnormal gene expression profile (Wood et al., 2007).
The increased oocyte yield counter-balances the reduced oocyte quality and the final result is similar clinical pregnancy and live birth rates between PCOS patients and controls following assisted reproduction (Bailey et al., 2014, Weghofer et al., 2007, Heijnen et al., 2006, Urman et al., 2004). A recent retrospective study reported decreased pregnancy and live birth rates in obese PCOS patients compared with lean PCOS patients (Bailey et al., 2014).
In vitro maturation (IVM) has recently become an alternative for standard assisted reproductive techniques, IVF and intracytoplasmatic sperm injection (ICSI) (Lindenberg, 2013). The main aim of this technique is to avoid the risk of ovarian hyperstimulation syndrome in PCOS, as the oocytes are retrieved from unstimulated cycles, but the success rates in terms of live birth are inferior to IVF (Das et al., 2014, Walls et al., 2015). Another concern regarding in vitro maturation, is the presence of high rates of meiotic abnormality in the immature oocytes, but no difference is observed in chromosomal and spindle configuration between PCOS and control oocytes (Zhu et al., 2015).